Willow bark EUROPEAN PHARMACOPOEIA 6.

01/2008:1583 the chromatogram obtained with test solution (b) the

corrected 6.0 zone due to salicin is clearly more intense and there
are, above the zone due to salicin, one (salicortin or
2′-O-acetylsalicortin, or possibly two tremulacin) faint
WILLOW BARK reddish-violet zones Other blue, yellow or brown zones
can occur in both chromatograms.
Salicis cortex TESTS
DEFINITION Foreign matter (2.8.2). Not more than 3 per cent of twigs
with a diameter greater than 10 mm, and not more than
Willow bark consists of the whole or fragmented dried bark
2 per cent of other foreign matter.
of young branches or whole dried pieces of current year twigs
of various species of genus Salix including S. purpurea L., Loss on drying (2.2.32). Not more than 11 per cent,
S. daphnoides Vill. and S. fragilis L. The drug contains not determined on 1.000 g of the powdered drug (355) (2.9.12),
less than 1.5 per cent of total salicylic derivatives, expressed by drying in an oven at 105 °C for 2 h.
as salicin (C13H18O7 ; Mr 286.3), calculated with reference to Total ash (2.4.16). Not more than 10 per cent.
the dried drug.
ASSAY
CHARACTERS
Examine by liquid chromatography (2.2.29), using
Markedly bitter. resorcinol R as the internal standard.
IDENTIFICATION Internal standard solution. Dissolve 50 mg of resorcinol R
in 10 ml of methanol R.
A. The bark is 1 mm to 2 mm thick and occurs in flexible,
elongated, quilled or curved pieces. The outer surface Test solution. To 0.5 g of the powdered drug (355) (2.9.12)
is smooth or slightly wrinkled longitudinally and add 50 ml of methanol R and heat under a reflux condenser
greenish-yellow to brownish-grey. The inner surface is for 30 min. Cool and filter. Take up the residue with 50 ml
smooth or finely striated longitudinally and white, pale of methanol R. Proceed as above. Combine the filtrates and
yellow or reddish-brown, depending on the species. The evaporate under reduced pressure. Take up the residue
fracture is short in the outer part and coarsely fibrous in with 5.0 ml of methanol R, add 5.0 ml of 0.1 M sodium
the inner region. The diameter of current year twigs is hydroxide and heat in a water-bath at about 60 °C under
not more than 10 mm. The wood is white or pale yellow. a reflux condenser, with frequent shaking for about 1 h.
After cooling, add 0.5 ml of 1 M hydrochloric acid. Dilute
B. Reduce to a powder (355) (2.9.12). The powder is pale the solution to 20.0 ml with a mixture of 50 volumes of
yellow, greenish-yellow or light brown. Examine under a methanol R and 50 volumes of water R. Add 1.0 ml of the
microscope using chloral hydrate solution R. The powder internal standard solution to 10.0 ml of this solution. Filter
shows : bundles of narrow fibres, up to about 600 µm through a membrane filter.
long, with very thick walls and surrounded by a crystal
Reference solution (a). Dissolve 18.5 mg of salicin R
sheath containing prism crystals of calcium oxalate ;
in 10.0 ml of a mixture of 20 volumes of water R and
parenchyma of the cortex with thick, pitted and deeply
80 volumes of methanol R and add 1.0 ml of the internal
beaded walls, and containing large cluster crystals of
standard solution.
calcium oxalate ; uniseriate medullary rays ; thickened and
suberised cork cells. Groups of brownish collenchyma Reference solution (b). Dissolve 1.0 mg of picein R in 1.0 ml
from the bud may be present. Twigs show, additionally, of reference solution (a).
fragments of lignified fibres and vessels from the xylem. The chromatographic procedure may be carried out using :
C. Examine by thin-layer chromatography (2.2.27), using a — a stainless steel column, 0.10 m long and 3 mm or 4 mm
TLC silica gel plate R. in internal diameter, packed with octadecylsilyl silica gel
Test solution (a). To 1.0 g of the powdered drug (500) for chromatography R (3 µm),
(2.9.12) add 20 ml of methanol R ; heat in a water-bath — as mobile phase at a flow rate of 1.0 ml/min a mixture
at about 50 °C, with frequent shaking, for 10 min. Cool of 1.8 volumes of tetrahydrofuran R and 98.2 volumes
and filter. of water R, containing 0.5 per cent V/V of phosphoric
Test solution (b). To 5.0 ml of test solution (a) add 1.0 ml acid R,
of a 50 g/l solution of anhydrous sodium carbonate R — as detector a spectrophotometer set at 270 nm,
and heat in a water-bath at about 60 °C for 10 min. Cool
and filter if necessary. — a loop injector.
Reference solution. Dissolve 2.0 mg of salicin R in 1.0 ml Inject 10 µl of reference solution (b). The assay is not valid
of methanol R. unless the resolution between the peaks corresponding to
salicin and picein and between the peaks corresponding to
Apply to the plate as bands 20 µl of each. Develop over a picein and resorcinol is at least 1.5.
path of 15 cm using a mixture of 8 volumes of water R,
15 volumes of methanol R and 77 volumes of ethyl Inject five times 10 µl of reference solution (a).
acetate R. Allow the plate to dry in air. Spray with a Inject three times 10 µl of the test solution. Continue the
mixture of 5 volumes of sulphuric acid R and 95 volumes chromatography for four times the retention time of the
of methanol R. Heat at 100 °C to 105 °C for 5 min peak corresponding to salicin.
and examine in daylight. The chromatogram obtained Calculate the percentage content of total salicylic derivatives,
with the reference solution shows in the middle third a expressed as salicin, from the expression :
reddish-violet zone due to salicin. In the chromatogram
obtained with test solution (a), the zone due to salicin
appears with only slight to moderate intensity. In

3220 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.0
S1 = area of the peak due to salicin in the
chromatogram obtained with the test solution,
S2 = area of the peak due to resorcinol in the
chromatogram obtained with the test solution,
S3 = area of the peak due to salicin in the chromatogram
obtained with reference solution (a),
S4 = area of the peak due to resorcinol in the
chromatogram obtained with reference
solution (a),
m1 = mass of the drug in the test solution, in milligrams,
m2 = mass of salicin in reference solution (a), in
milligrams,
p the same solvent.
= percentage content of salicin in the reference
substance
01/2008:0593
corrected 6.0
WOOL ALCOHOLS
Alcoholes adipis lanae
DEFINITION
Mixture of sterols and higher aliphatic alcohols from
wool fat. It may contain not more than 200 ppm of
butylhydroxytoluene.
Content : minimum 30.0 per cent of cholesterol.
CHARACTERS
Appearance : pale-yellow or brownish-yellow, brittle mass
becoming plastic on heating.
Solubility : practically insoluble in water, soluble in
methylene chloride and in boiling ethanol, slightly soluble
in alcohol (90 per cent V/V).
IDENTIFICATION
Dissolve 50 mg in 5 ml of methylene chloride R and add
1 ml of acetic anhydride R and 0.1 ml of sulphuric acid R.
Within a few seconds, a green colour develops.
TESTS
Appearance of solution. To 1.0 g add 10 ml of light
petroleum R1 and shake while warming in a water-bath. The
substance dissolves completely. After cooling, the solution is
clear (2.2.1).
Alkalinity. Dissolve 2.0 g in 25 ml of hot alcohol (90 per
cent V/V) R and add 0.5 ml of phenolphthalein solution R1.
No red colour develops.
Melting point (2.2.15) : minimum 58 °C.
Melt the substance to be examined by heating in a water-bath
at a temperature which exceeds the expected melting point
by not more than 10 °C ; introduce the substance to be
examined into the capillary tubes and allow to stand at
15-17 °C for not less than 16 h.
Acid value (2.5.1) : maximum 2.0.
If necessary, heat in a water-bath under a reflux condenser
to dissolve the substance to be examined.
Hydroxyl value (2.5.3, Method A) : 120 to 180.
Peroxide value (2.5.5, Method A) : maximum 15.
Take from the substance to be examined wedge-shaped
pieces whose base consists of part of the surface. Melt the
General Notices (1) apply to all monographs and other texts
Wool alcohols
pieces before carrying out the determination. Before adding
0.5 ml of saturated potassium iodide solution R, cool the
solution obtained to room temperature.
Saponification value (2.5.6) : maximum 12.0, determined on
2.00 g. Heat under reflux for 4 h.
Butylhydroxytoluene. Gas chromatography (2.2.28).
Internal standard solution. Dissolve 0.20 g of methyl
decanoate R in carbon disulphide R and dilute to 100.0 ml
with the same solvent. Dilute 1.0 ml of the solution to
10.0 ml with carbon disulphide R.
Test solution (a). Dissolve 1.0 g of the substance to be
examined in carbon disulphide R and dilute to 10.0 ml with
Test solution (b). Dissolve 1.0 g of the substance to be
examined in carbon disulphide R, add 1.0 ml of the
internal standard solution and dilute to 10.0 ml with carbon
disulphide R.
Reference solution. Dissolve 0.20 g of butylhydroxy-
toluene R in carbon disulphide R and dilute to 100.0 ml with
the same solvent. Dilute 1.0 ml of the solution to 10.0 ml
with carbon disulphide R. To 1.0 ml of this solution add
1.0 ml of the internal standard solution and dilute to 10.0 ml
with carbon disulphide R.
Precolumn : column filled with silanised glass wool.
Column :
— size : l = 1.5 m, Ø = 4 mm,
— stationary phase : silanised diatomaceous earth for gas
chromatography R impregnated with 10 per cent m/m of
poly(dimethyl)siloxane R.
Carrier gas : nitrogen for chromatography R.
Flow rate : 40 ml/min.
Temperature :
— column : 150 °C,
— injection port : 180 °C,
— detector : 300 °C.
Detection : flame ionisation.
Limit :
— butylhydroxytoluene : maximum 200 ppm.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 2.000 g by drying in an oven at 105 °C.
Total ash (2.4.16) : maximum 0.1 per cent.
Water-absorption capacity. Place 0.6 g of the substance to
be examined and 9.4 g of white soft paraffin R in a mortar
and melt on a water-bath. Allow to cool and incorporate
20 ml of water R, added in portions. Within 24 h no water is
released from the almost white, ointment-like emulsion.
ASSAY
Dissolve 0.1000 g in 12 ml of hot alcohol (90 per cent V/V) R.
Allow to stand for 18 h, filter through a sintered-glass filter
(16) (2.1.2) and wash the filter with 2 quantities, each of
15 ml, of alcohol (90 per cent V/V) R. Combine the filtrate
and washings, add 20 ml of a freshly prepared 10 g/l solution
of digitonin R in alcohol (90 per cent V/V) R and heat to
about 60 °C. Allow to cool, filter through a sintered-glass
filter (16) (2.1.2), wash the residue with 10 ml of alcohol
(90 per cent V/V) R and dry to constant mass at 100-105 °C.
1 g of residue is equivalent to 0.239 g of cholesterol.
STORAGE
In a well-filled container, protected from light.
3221